Mapping the Nonhistone Interactome of the TRIM24 Bromodomain Using Genetically Encoded Complementary Photocrosslinkers

TRIM24 is a tandem epigenetic reader containing plant homeodomain (PHD) and bromodomain modules in its C-terminal region. The PHD domain recognizes unmodified lysine 4 of histone H3, whereas the bromodomain recognizes acetylated histones H3 and H4. However, whether the TRIM24 bromodomain also engages acetylated nonhistone proteins remains poorly understood. To address this question, we developed site-specific photocrosslinking probes for the TRIM24 bromodomain using interaction-based protein profiling (IBPP). 4-azido-L-phenylalanine (AzF) and 4-benzoyl-L-phenylalanine (BzF) were genetically incorporated at L922, a residue positioned within the acetyllysine-binding pocket. Both variants preserved recognition of acetylated histone peptides and underwent efficient UV-dependent photocrosslinking. Quantitative proteomics of photocaptured proteins revealed distinct profiles for the two probes, identifying NPM1 among the L922AzF-enriched proteins and CDK9 among the L922BzF-enriched proteins. Additional candidate proteins included MAP4, RAN, and SND1. ChIP-seq analysis showed overlapping genomic enrichment of TRIM24, CDK9, and NPM1 near transcription start sites of cancer-associated genes. KEGG analysis further placed the identified proteins in several signalling pathways. Collectively, this study demonstrates that site-specific photocrosslinking can expand interactome analysis of epigenetic reader domains and provides candidate nonhistone proteins associated with the TRIM24 bromodomain for further investigation.